Molecular Neurobiology

MicroRNA-455-3p is one of the highly conserved miRNAs involved in several human diseases but newly explored by our lab in Alzheimers disease (AD). Our past studies unveiled the biomarker and therapeutic potentials of miR-455-3p in AD. Our in vitro study exhibited the protective role of miR-455-3p against AD toxicities in reducing full-length APP and amyloid-{beta} (A{beta}) protein levels, and also reducing defective mitochondrial biogenesis, impaired mitochondrial dynamics and synaptic deficiencies. Next, we sought to determine the essential roles of miR-455-3p in AD using mouse models. Therefore, for the first time we generated both transgenic (TG) and knockout (KO) mouse models of miR-455-3p. We determined the positive and negative effects of miR-455-3p on mice cognitive function, mitochondrial biogenesis, mitochondrial dynamics, mitochondrial number & length, dendritic spine density, synapse numbers and synaptic activity in 12-month-old miR-455-3p TG and KO mice. MiR-455-3p TG mice lived 5 months longer than wild-type (WT) mice, whereas KO mice lived 4 months shorter than their WT counter parts. Morris water maze test showed improved cognitive behavior, spatial learning and memory in miR-455-3p TG mice relative to age-matched WT mice and miR-455-3p KO mice. Further, mitochondrial biogenesis, dynamics and synaptic activities were enhanced in miR-455-3p TG mice, while these were reduced in KO mice. Overall, miR-455-3p TG mice displayed protective effects and miR-455-3p KO mice exhibited deleterious effects in relation to AD pathogenesis. Both mouse models could be ideal research tools to understand the molecular mechanism of miR-455-3p in AD and other human diseases.

BackgroundE3 ubiquitin ligases, integral components of the proteasomal degradation cascade, are critical for regulating the cellular proteome via canonical proteasome-mediated protein degradation; however, the non-canonical functions of these ligases in neuronal development are poorly understood. Our study focuses on a special class of E3 ubiquitin ligases known as RNA Binding Ubiquitin Ligases (RBUL) that harbour RNA-binding domains; allowing them to acquire all the properties of RNA-binding proteins (RBPs) and regulate transcriptional or post-transcriptional changes associated with the control of gene expression in cellular phenotypes. We aim to identify one such RUBL in the context of the highly dynamic yet stringently controlled process of neural proliferation and neural fate determination. ResultsMEX3B protein is a member of the MEX3 family and a part of the RBUL class of E3 ligases. It is differentially expressed in Neural Progenitor Cells (NPCs) upon differentiation. Mex3b RNA and protein were found to have temporally opposing expression patterns in presence of basic fibroblast growth factor (bFGF), a key signalling protein involved in neuronal proliferation. MEX3B is required for maintenance of the proliferative state of NPCs, whereas, its knockdown promotes transition from proliferative to differentiation state even in presence of bFGF that restricts differentiation. Furthermore, the knockdown of MEX3B protein results in the appearance of morphological hallmarks associated with early stages of neuronal differentiation including increase in neurite length and complexity. MEX3B interacts with the pro-proliferative transcription activator REST and the long non-coding RNA, HOTAIR. The study suggests the existence of a bFGF-dependent, combinatorial axis involving Mex3b, REST and HOTAIR, for the maintenance of NPC proliferative states. ConclusionMEX3B, containing RNA binding motifs, is a unique E3 ligase that is necessary for bFGF-dependent proliferation. Mex3b protein invokes its non-canonical function of an RNA binding protein to form a tripartite complex with the transcription activator REST and HOTAIR lncRNA to define the proliferative state of NPCs. The study highlights a unique feature of special E3 ligases in neuronal proliferation during brain development that was previously overlooked.

The neurotrophin brain-derived neurotrophic factor (BDNF) plays a key role in neuronal development and synaptic plasticity. The discovery that BDNF mRNA can be transported in neuronal dendrites in an activity-dependent manner has suggested that its local translation may support synapse maturation and plasticity. However, a clear demonstration that BDNF mRNA is locally transported and translated at activated synapses in response to long-term potentiation (LTP) is still lacking. Here, we study the dynamics of BDNF mRNA dendritic trafficking following induction of chemical-LTP (cLTP). Dendritic transport of BDNF transcripts was analysed using the MS2 system for mRNA visualization, and chimeric BDNF-GFP constructs were used to monitor protein synthesis in living neurons. We found that within 15 following cLTP induction, most BDNF mRNA granules become stationary and transiently accumulate in the dendritic shaft at the basis of the spines similarly to the control CamkII mRNA which increased also inside the spines, at 60 post-cLTP. At 60 but not at 15 from cLTP induction, we observed an increase in BDNF protein levels within the spine. Taken together, these findings suggest that BDNF mRNA trafficking is arrested in the early phase of cLTP, providing a local source of mRNA for translation of BDNF at the basis of the spine followed in the late LTP phase, by translocation of the BDNF protein within the spine head. StatementBrain-derived neurotrophic factor (BDNF) plays a key role in neuronal development and synaptic plasticity. In this study, we investigate two unresolved questions in neuronal plasticity: a) whether the post-synaptically released BDNF can be locally synthesized in this compartment, and b) whether the local translation of BDNF occurs in dendrites, or within the spine. Using chimeric constructs ectopically expressed in living primary hippocampal neurons, we tracked BDNF mRNA trafficking within the dendrites and its local translation following induction of chemical-LTP (cLTP) by forskolin. We show that in the early phase of cLTP induction (15), BDNF mRNA becomes confined at the basis of the spines providing a local source for translation of the protein followed in the late LTP phase (60), by translocation of the BDNF protein within the spine head.

N6-methyladenosine (m6A) is the most abundant and conserved transcriptional modification in eukaryotic RNA, regulating RNA fate. While the functions of m6A in the development of the mammalian brain have been extensively studied, its roles in synaptic plasticity, cognitive decline, motor function, or other brain circuits remain underexplored. To date, the role of this modification in Parkinsons disease (PD) and other synucleinopathies has been largely unknown. Here, we investigated the m6A epitranscriptome in a mouse model of synucleinopathy. We performed m6A RNA immunoprecipitation sequencing (meRIP-seq) to obtain the m6A epitranscriptome of the midbrain in young (3 mo) and aged (15 mo) A30P-aSyn transgenic mice (aSyn Tg) and C57BL6 control wild type (Wt) mice. We observed hypermethylation of synaptic genes in 3 mo aSyn Tg mice compared to age-matched Wt mice. This methylation was reduced during ageing, with synaptic genes becoming increasingly hypomethylated. Using immunofluorescence imaging alongside biochemical analysis, we further investigated the expression of m6A regulatory enzymes -- writer, N6-Adenosine-Methyltransferase Complex Catalytic Subunit (METTL3); reader, YTH N6-methyladenosine RNA-binding protein (YTHDF1); and eraser, fat mass and obesity-associated protein (FTO) -- in the cortex, striatum, hippocampus, and cerebellum of Wt and aSyn Tg mice, as well as in primary cortical neuronal cultures. We observed that the levels of METTL3, YTHDF1 and FTO were similar between Wt and aSyn Tg mice. Interestingly, the writer protein METTL3 was found in both the nucleus and in the post-synaptic compartment in neuronal cultures. Our findings suggest that alterations in the regulation of m6A RNA methylation may be associated with neurodegeneration and ageing and that this level of epitranscriptomic regulation plays a significant role at the synapse.

In the central nervous system (CNS), axons and its surrounding myelin sheaths, generated by oligodendrocytes, greatly depend on each other, where oligodendrocytes provide axons with both trophic and metabolic support. Across spices, assessment of the axon-myelin ultrastructure is the key-approach to visualize de- and re-myelination of axons. However, this assessment omits to provide information on axonal homeostasis or how axon-myelin influence one another. Since mitochondria may adjust in size thus mirroring the intracellular physiological and metabolic status we applied this to myelinated axons in the CNS. We herein show that a large axonal mitochondria diameter correlates with thinner surrounding myelin sheaths across different CNS tracts and species, including human. We also show that the relation between axonal mitochondria diameter and surrounding myelin thickness is a valuable measurement to verify advanced remyelination in two commonly used experimental demyelinating models, namely the cuprizone and the lysolecithin (LPC) model. Lastly, we show that axonal mitochondria adjust in diameter in response to the thickness of the axonal surrounding myelin whereas the opposite adaption was absent. In summary, the link between axonal mitochondria diameter and surrounding myelin thickness provide insight on the axon-myelin relation both during homeostasis and pathological conditions. This link is also translational applicable and can thus contribute to a better understanding on how to study remyelination using experimental models.

Oligodendrocytes (OLs) are vital for myelin formation in the Central Nervous System. OLs can be produced by the maturation of oligodendrocyte precursor cells (OPCs) present throughout the brain parenchyma or from the differentiation of subventricular zone-derived neural stem cells (SVZ-NSCs). Importantly, efficient differentiation from SVZ-NSCs remains a significant area of research due to its potential for remyelination in demyelinating disorders. In this work, we studied the role of brain-derived neurotrophic factor (BDNF) and adenosine A2A receptors (A2ARs), as well as the putative crosstalk between these two modulatory mechanisms, in regulating oligodendrogenesis from SVZ-NSCs. Using a neurosphere culture system, we observed that BDNF significantly increased the mRNA expression levels of OPC cell markers after 2 days in vitro (DIV), an effect blocked by the A2AR antagonist ZM 241385. This early transcriptional regulation by BDNF was followed by changes in the percentage of both OPCs and mature OLs in culture at DIV 7 and 14. Interestingly, blocking A2ARs prevented the potentiating effect of BDNF on the percentage of OLs at DIV 14. Concerning the morphology of mature OLs, BDNF influenced their maturation by reducing branching near the soma at DIV 7, an effect that was not observed at 14 DIV, when all treatments resulted in similar OL morphology. Overall, our results establish BDNF as a regulator of OL formation from SVZ-NSCs, with A2AR-BDNF interaction modulating the differentiation process.

Dysregulated mammalian target of rapamycin (mTOR) activity is associated with various neurodevelopmental disorders ranging from idiopathic autism spectrum disorders to syndromes caused by single gene defects. This suggests that maintaining mTOR activity levels in a physiological range is essential for brain development and functioning. Upon activation, mTOR regulates a variety of cellular processes such as cell growth, autophagy and metabolism. On a molecular level, however, the consequences of mTOR activation in the brain are not well understood. Low levels of cholesterol are associated with a wide variety of neurodevelopmental disorders. We here describe numerous genes of the sterol/cholesterol biosynthesis pathway to be transcriptionally regulated by mTOR complex 1 (mTORC1) signaling in vitro in primary neurons and in vivo in the developing cerebral cortex of the mouse. We find that these genes are shared targets of the transcription factors SREBP, SP1 and NF-Y. Prenatal as well as postnatal mTORC1 inhibition downregulated expression of these genes which directly translated into reduced cholesterol levels pointing towards a substantial metabolic function of the mTORC1 signaling cascade. Altogether, our results indicate that mTORC1 is an essential transcriptional regulator of the expression of sterol/cholesterol biosynthesis genes in the developing brain. Altered expression of these genes may be an important factor contributing to the pathogenesis of neurodevelopmental disorders associated with dysregulated mTOR signaling.

N-methyl-D-aspartate receptors (NMDAR) are downregulated in schizophrenia possibly through microRNAs (miRNAs) that are differentially expressed in this condition. We screened the miRNAs that are altered in schizophrenia against the targets, Grin2A and Grin2B subunits of NMDAR using bioinformatic tools. Among the predicted miRNAs some interacted with the 3-UTR sequences of Grin2A (miR-296, miR-148b, miR-129-2, miR-137) and Grin2B (miR-296, miR-148b, miR-129-2, miR-223) in dual luciferase assays. This was supported by downregulation of the GluN2B protein in primary hippocampal neurons upon overexpressing Grin2B targeting miRNAs. In two models of schizophrenia-pharmacological MK-801 model and neurodevelopmental methylazoxymethanol acetate (MAM) model which showed cognitive deficits - protein levels of GluN2A and GluN2B were downregulated but their transcript levels were upregulated. MiR-296-3p, miR-148b-5p and miR-137 levels showed upregulation in both models which could have interacted with Grin2A/Grin2B transcripts resulting in translational arrest. In MAM model, reciprocal changes in the expression of the 3p and 5p forms of miR-148b and miR-137 were observed. Expression of neuregulin 1 (NRG1), BDNF and CaMKII, genes implicated in schizophrenia, were also altered in these models. This is the first report of downregulation of GluN2A and GluN2B by miR-296, miR-148b and miR-129-2. Mining miRNAs regulating NMDA receptors might give insights into the pathophysiology of this disorder, providing avenues in therapeutics.

Hypoxia, a condition of inadequate oxygen supply, is a common phenomenon affecting neurons and brain tissue, leading to significant implications for neuronal health and function. The prevalence of hypoxia in the brain is associated with various neurological conditions, making it a critical area of study. Neuritogenesis, the process of neurite outgrowth, is an essential aspect of neuronal development and connectivity and is particularly sensitive to hypoxic stress. Investigating how hypoxia affects neurite outgrowth is vital for understanding neuronal response and adaptation under low oxygen conditions. This study explores how hypoxic stress affects neurite regulation mediated by Contactin Associated Protein-1 (Caspr1) in primary mouse embryonic cortical neurons. Hypoxia, induced by culturing neurons in a 2% oxygen environment, significantly reduced neurite length and induced notable changes in growth cone morphology. Concurrently, we observed an upregulation in the expression of Caspr1 and its transcriptional regulator C/EBP, suggesting a compensatory role for Caspr1 in neurite extension under low oxygen conditions. Shorter hypoxia exposure periods revealed a dynamic biphasic response in Caspr1 levels, with an initial decrease followed by a substantial increase, correlating with corresponding changes in neurite length. This pattern emphasizes the critical involvement of Caspr1 in adapting neurite growth to fluctuating hypoxia duration. Furthermore, comparative analyses using wild-type and Caspr1 knockout Neuro2a cells demonstrated that the absence of Caspr1 mitigates hypoxia-induced neurite shortening, indicating a potential protective role against hypoxic stress. Additionally, hypoxia profoundly impacted mitochondrial morphology and function. Under hypoxic conditions, mitochondria transitioned to a more spherical shape. Mitochondrial respiration analysis revealed significant reductions in oxygen consumption rates (OCR), highlighting compromised mitochondrial function during hypoxia. These findings underscore the multifaceted role of Caspr1 in neurite regulation and mitochondrial adaptation to hypoxic stress. The study provides insights into the molecular mechanisms underpinning hypoxia-induced changes in neuronal morphology and function. Understanding these processes opens avenues for therapeutic strategies targeting Caspr1 in treating neurological disorders characterized by hypoxic stress. Future research will benefit from extending these investigations to more complex models, such as brain organoids, to further elucidate the metabolic and structural changes under hypoxia and their implications for neurodegenerative diseases.

The lipid signalling molecule prostaglandin E2 (PGE2) is important in healthy brain development. Abnormal PGE2 levels during prenatal development, which can be influenced by genetic causes and exposure to various environmental risk factors, have been linked to increased prevalence of Autism Spectrum Disorders (ASDs). Growing research in animal models aims to provide evidence for the mechanisms by which increased or reduced PGE2 levels influence brain development. In this study, we show that maternal exposure to PGE2 in mice at gestational day 11 (G11) results in molecular changes within the cerebellum and associated behaviours in offspring. We observed a decrease in cerebellar cell density originating at G11 (in males and females) and at G16 (in females only). In Golgi-COX-stained cerebellar slices from PGE2-exposed offspring at the postnatal day 30 (PN30), we found an increase in dendritic arborization, the odds of observing dendritic loops, dendritic spine density, and the odds of observing mature (mushroom-shaped) spines. We also observed a decrease in the expression level of the cytoskeletal protein {beta}-actin, the actin associated protein spinophilin, and the cell adhesion protein N-Cadherin. In addition, we found that specifically PGE2-exposed male offspring exhibited abnormal cerebellar related motor function. This study adds further evidence that changes in the PGE2 levels during critical times may impact the developing brain differently in males and females. These findings also emphasize the importance of examining sex differences in research relevant to neurodevelopmental disorders.

Several genome-wide association studies have associated CACNA1C variants with psychiatric disorders. The molecular mechanisms involved are poorly understood. Taking advantage of the zebrafish larva as a model, we investigated how two different mutations in cacna1c - sa10930 (nonsense mutation) and sa15296 (splice site mutation), affect neuronal function. We characterized changes in cacna1c mRNA, neurotransmitter levels and behaviour, as well as whole-brain activity using single electrode local field potential recordings. Both point mutations resulted in a significant reduction in cacna1c mRNA, as well as social behaviour and prepulse inhibition deficits. Whereas sa15296 mutants displayed abnormal locomotor and open-field behaviour, we observed normal behaviour in the sa10930 mutants. Brain recordings from both mutants had lower spectral power while sa15296 displayed significant seizure-like activity. Finally, sa10930 homozygotes showed increased dopamine and serotonin levels, decreased gamma-aminobutyric acid (GABA) levels, and unchanged glutamate levels while homozygous sa15296 larvae showed increased levels of serotonin and glutamate, and unaffected levels of GABA and dopamine. Our work provides new insights into the functional role of CACNA1C in behavioural, electrophysiological and biochemical traits linked to psychiatric disorders. We show a functional role for the non-coding mutation (sa15296) in the cacna1c in vivo animal model. Consistent with existing hypotheses, our data suggest that disruption of gene expression, neurotransmission, and cortical excitability are involved in CACNA1C-related mechanisms of psychiatric disorders.

Synapse dysfunction is the root cause of Alzheimers disease (AD). Uninterrupted and regulated synapse action is crucial to maintain healthy brain function. Our previous study discovered microRNA-502-3p (miR-502-3p), a synapse-specific miRNA, highly expressed at the AD synapses. Further, in vitro studies unveiled the biological relevance of miR-502-3p in modulating GABA receptor function, synaptic activity and mitochondrial morphology. Current study focuses to investigate the role of miR-502-3p in vivo using stereotaxic injection of miR-502-3p overexpression (OE) and suppression (sponge) lentivirus (LV) into the hippocampus of C57BL/6 wild-type (WT) mice. MiR-502-3p OE and sponge LV were characterized by transducing HT22 cells followed by QRT-PCR and miRNAScope analysis of miR-502-3p. MiR-502-3p OE LV showed a very high-fold upregulation and sponge LV showed significant reduction in miR-502-3p levels. MiR-502-3p OE and sponge LV were injected into three months old WT mice brain hippocampus. Overexpression and suppression effects of miR-502-3p were studied on synaptic proteins, synapse number, mitochondrial morphology and dendritic spine density at eight-weeks post-injection. Mice injected with miR-502-3p OE LV showed reduced levels of synaptic proteins, diminished synapse formation, defective mitochondrial morphology and reduced dendritic spine density relative to control LV treated mice. While mice treated with sponge LV showed elevated levels of synaptic proteins, augmented synapses, improved mitochondrial morphology and elongated dendrites and spine density. Our in vivo study unveiled translational abilities of miR-502-3p to restore synapse dysfunction in AD and other neurological disorders.

This study explored the neurogenic and microglial modulatory properties of botulinum toxin in the hippocampus of aging experimental mice. Therapeutic botulinum toxin (BoNT) treatment is widely practiced to reduce the excessive discharge of acetylcholine (ACh) in the management of aging and neurological deficits. While the production of new neurons in the adult brain contributes to cognitive functions, age-related diseases with excessive release of ACh and progressive neuroinflammation have been characterized by impaired hippocampal neurogenesis and memory loss. Therefore, we investigated the effect of BoNT on the regulation of hippocampal neurogenesis, focusing on doublecortin (DCX)-positive immature neurons in the hippocampus of aging experimental mice. We also assessed ionized calcium-binding adapter molecule 1 (Iba1)-positive microglia and the expression of cyclooxygenase (COX)-2, a key inflammatory response element, using reverse transcription polymerase chain reaction (RT-PCR). Results revealed a prominent increase in DCX-positive cells in the BoNT-treated animals compared to the control group. Additionally, the reduced number of microglia accompanied by decreased mRNA expression of COX-2 was evident in the BoNT-treated animals. These dual effects suggest that BoNT could be a promising therapeutic agent for mitigating age-related neuroregenerative decline and neuroinflammation responsible for cognitive impairments.

The Mas-related G protein-coupled receptor D (MrgD) is primarily known for its role in peripheral nociception and, more recently, as a receptor for alamandine, influencing cardiovascular function and exhibiting antidepressant-like effects. However, its function within the central nervous system, particularly in motor and reward-related circuits, remains largely unexplored. Here, we investigate the role of MrgD in the nigrostriatal pathway using proteomic approaches focused on post-translational modifications in MrgD-knockout (KO) mice. Integrated proteomic, phosphoproteomic, and N-glycoproteomic analyses revealed significant alterations in synaptic vesicle-associated proteins, pointing to impaired neurotransmission in the nigrostriatal system of KO mice. These molecular findings were supported by neurotransmitter quantification and functional assays demonstrating impaired synaptic vesicle exocytosis. Pharmacodynamic analyses showed that MrgD modulates synaptic exocytosis in an agonist-selective manner, being responsive to alamandine but not {beta}-alanine. Furthermore, behavioral analyses revealed increased locomotor activity and compulsive-like behavior in MrgD-deficient mice, without impairments in short- or long-term memory. Together, these findings uncover a new role for MrgD beyond its involvement in nociception, highlighting this receptor as a potential therapeutic target for neurological disorders involving motor hyperactivity and compulsivity.

Kv1 voltage-gated potassium channels determine key functional neuronal properties. Their activity is modulated by subunit composition and post-translational modifications such as phosphorylation and glycosylation. Using an antibody directed against a phosphotyrosine (Y458) located in the C-terminal tail of Kv1.2, we identified yet unreported high molecular weight forms of Kv1.2 among them, a phosphorylated and heavily glycosylated 100 kDa form. Owing to the significant downregulation of Kv1.2 in LGI1-dependent autosomal dominant lateral temporal lobe epilepsy, we investigated, in total brain and the hippocampal formation of both WT and Lgi1-/- mice, the distribution of phosphoY458 Kv1.2 and we compared their respective proteomic interactomes with those of Kv1.2. In addition to major differences between the interactomes of pY458Kv1.2 and Kv1.2 in WT and Lgi1-/-, we found a major reshaping of pY458 Kv1.2 molecular neighbourhood between WT and Lgi1-/- as well as a significant upregulation of the glycosylated form in Lgi1-/-.

Microglia and astrocytes are essential in sustaining physiological networks on the central nervous system, with their ability to remodel the extracellular matrix, being pivotal for synapse plasticity. Recent findings challenge the traditional view of homogenous glial populations in the brain, uncovering morphological, functional and molecular heterogeneity among glial cells. This diversity has significant implications for both physiological and pathological brain states. In the present study, we mechanically induced a Schaffer collateral lesion (SCL) in mouse enthorino-hippocampal slice cultures to investigate glial behavior, i.e., microglia and astrocytes, under metalloproteinases (MMPs) modulation in the lesioned area, CA3, and the denervated region, CA1. We observed distinct response patterns in microglia and astrocytes 3 days after the lesion. Notably, GFAP-expressing astrocytes showed no immediate changes post-SCL. Microglia responses varied depending on their anatomical location. The MMPs inhibitor GM6001 did not affect microglial reactions in CA3, while increasing the Iba1 cells numbers in CA1, underscoring the complexity of the hippocampal neuroglial network post-injury. These findings highlight the importance of understanding glial regionalization following neural injury and MMPs modulation, and pave the way for further research into glia-targeted therapeutic strategies for neurodegenerative disorders.

Neuronal polarization is a fundamental process in the formation of functional neural circuits, relying on the precise coordination between cytoskeletal regulatory signals and mechanisms that sustain cellular integrity. Disruption of these processes compromises neuronal differentiation and survival, and various neurotoxic compounds, including certain pesticides, have been associated with such dysfunctions. In this context, identifying molecules that counteract these detrimental effects is of significant therapeutic interest. Neuronal polarization is essential for the establishment of functional neural circuits and relies on coordinated regulation of actin cytoskeleton dynamics, RhoA/ROCK signaling, and mitochondrial function. Here, we investigated the neuroprotective and neurorestorative potential of the ganglioside GM1 and its oligosaccharide derivative, osGM1, in primary hippocampal pyramidal neurons exposed to the mitochondrial neurotoxin rotenone. Rotenone induced a marked arrest of neuronal development, impaired axonal elongation, and disrupted mitochondrial organization and membrane potential. Both GM1 and osGM1 promoted recovery of neuronal polarity and axonal growth, exerting protective and restorative effects even under continuous toxin exposure, with osGM1 showing superior efficacy. Notably, osGM1 also reversed axonal growth deficits caused by pathological actin stabilization. Mechanistically, osGM1 normalized rotenone-induced hyperactivation of the RhoA/ROCK pathway without altering basal signaling and partially restored mitochondrial network integrity and function. Collectively, these findings identify osGM1 as a multi-target modulator of cytoskeletal and mitochondrial dysfunction and support its translational potential as a therapeutic strategy to counteract neurotoxin-induced neuronal damage.

Microglia play a critical role in central nervous system (CNS) pathologies including multiple sclerosis (MS), and their modulation offers therapeutic potential especially during progressive disease courses. Using cell culture and experimental autoimmune encephalomyelitis (EAE) models, we investigated microglial dynamics during depletion and repopulation (MGrepo) and their modulation using siponimod (sipo), an established CNS penetrating MS medication. Repopulating microglia exhibited a transient reactive state (CD86, MHC-II, Il1b, Tnf). Sipo modulated microglia populations, increasing CD163+, CD206+, and CX3CR1+ while reducing CD86+MHC-II+ cells accompanied by a reduction of neuronal damage. Proteomic spinal cord analysis revealed protein expression alterations by MGrepo and sipo linked to inflammation, myelination, and neuronal structural organization, supported by RNA sequencing of the spinal cord. The neuroinflammation attenuating role of sipo could be linked to cell maintenance and myelin formation associated processes. These findings highlight the capacity of pharmacological interventions to modulate microglia, offering new insights into therapeutic strategies targeting microglial activity in neuroinflammatory diseases.

White matter abnormalities are a hallmark of cerebral small vessel disease (cSVD) and are closely linked to cognitive decline and dementia. However, despite their clinical importance, underlying mechanisms remain poorly understood. Collagen IV, encoded by genes COL4A1/COL4A2, is a major component of the basement membrane, a specialised extracellular matrix (ECM) structure. Mutations in these genes cause a genetic form of cSVD. We tested the hypothesis that ECM defects caused by a Col4a1 mutation lead to white matter pathology using an established mouse model of cSVD (Col4a1+/Svc). Behavioural testing with magnetic resonance diffusion tensor imaging, pathology and ultrastructural investigations of white matter were studied. The studies revealed that Col4a1+/Svc mice have cognitive impairments, reduced myelinating oligodendrocyte pools, axonal myelination defects, and altered white matter structural integrity. Proteomic analysis, of isolated white matter from Col4a1+/Svcmice, identified extensive changes to ECM composition and endoplasmic reticulum (ER) biology including ER stress induction. We also demonstrated that targeting protein folding to promote collagen secretion and reduce ER stress, increased myelinating oligodendrocytes and axon-glial integrity in Col4a1+/Svc mice. These data provide novel insight into the pathomolecular mechanisms of white matter abnormalities in cSVD and identify a modifiable pathway as a putative therapeutic target for cSVD.

Metachromatic leukodystrophy (MLD) is a lysosomal storage disorder typically resulting from biallelic loss-of-function variants in the ARSA gene which encodes the lysosomal enzyme, arylsulphatase A, leading to the accumulation of its substrate, sulphatide, and widespread demyelination. Although gene therapy is available for MLD, it is limited by high cost and a narrow window for intervention, which means the development of therapies for MLD remains a key goal. The aim of the present study was to explore disease mechanisms in MLD with a view to identifying novel targets for therapeutic intervention for patients who cannot avail of gene therapy. Postmortem globus pallidus and dentate nucleus tissue was obtained from MLD cases (N=5; age 2-33 years old) and compared to age-, sex and ethnicity matched controls (N=5) and studied using discovery proteomics which demonstrated a marked inflammatory response, activation of the mTOR pathway, oxidative stress and metabolic remodelling in MLD cases. Histological analysis of inflammatory markers, including the terminal fragment of complement pathway activation, C3d, and the secreted glycoprotein YKL-40, a commonly used biomarker for inflammation, demonstrated their enrichment in MLD cases. Given that the mTOR pathway plays a key role in supressing autophagy, we next investigated autophagy and identified the accumulation of autophagosomes in MLD cases, consistent with deficient autophagy. Taken together, these findings suggest inflammation and autophagy dysfunction are key processes involved in MLD and that the mTOR pathway could be a novel therapeutic target for MLD.